Proteolysis by the ubiquitin (Ub) system plays essential roles in a multitude of biological processes, and has major ramifications for human health. Studies of the Ub-dependent N-end rule pathway by this laboratory over the last three decades were made possible, to a large extent, by the present grant (GM031530), currently in its 28th year of supporting our research. A focus of this renewal application stems from the 2010 discovery by the lab that the N-terminal acetylation (Nt-acetylation) of cellular proteins creates specific N-terminal degradation signals (degrons), termed AcN-degrons. The resulting expansion of the N-end rule pathway necessitated the change of the earlier title of this grant (The Functions, Mechanisms, and Regulation of N-Terminal Arginylation) to its present title, N-Terminal Acetylation and Protein Degradation by the N-End Rule Pathway. Specific Aims (described more briefly than in Research Plan): 1) Studies of the Nt-acetylation branch of the N-end rule pathway in the yeast Saccharomyces cerevisiae that include the use of recent mass spectrometry-based technologies to identify specific physiological substrates containing AcN-degrons. 2) Further development of the Ub-Reference Technique (URT), a method introduced by this laboratory in 1996. A novel (unpublished) version of URT t allows a nascent protein to undergo modifications (including Nt-acetylation) without kinetic delays. In another project of this Aim, we employ a different technique, based on activation of dormant N-degrons, to tackle a physiologically important problem of the posttranslational (as distinguished from cotranslational) Nt-acetylation. 3) Studies of the Nt-acetylation branch of the N-end rule pathway in mammalian cells. Proteins containing AcN-degrons have not been identified in mammals, until now. We shall focus, initially, on two proteins: Rgs2, a regulator of specific G proteins; and Aanat, the serotonin N-acetyltransferase. Aanat produces Ac-serotonin, the immediate precursor of melatonin, a hormone that regulates circadian rhythm. Regulated degradation of Aanat is likely to be mediated by its AcN-degron. 4) Studies of the previously known part of the N-end rule pathway, now called the UBR/arginylation branch. One of these projects is a study of Atl1 (Ate1's ligand-1) and its complex with Arg-transferase, Ate1. Other studies shall explore the recent (also unpublished) discovery that the UBR branch of the N-end rule pathway is mediated by a physical complex between Ubr1 (the previously known E3 of this pathway) and Ufd4, the E3 of the UFD (Ub-Fusion-Degradation) pathway. Until recently, the UFD pathway, identified and studied by this laboratory in 1986-1995, was thought to be entirely distinct from the N-end rule pathway.